The outer membranes of Gram-negative bacteria are barriers to diffusion of certain nutrients into the periplasmic space and subsequent transport into the cell. To acquire large, scarce, important nutrients such as iron-siderophores and vitamin B12, active transporters with sub-nanomolar affinities for their transport substrates are located in the outer membrane. Because the outer membrane lacks a conventional energy source, energy for active transport of iron-siderophores and vitamin B12 across the outer membrane and into the periplasmic space derives from the protonmotive force of the cytoplasmic membrane. The TonB-ExbB-ExbD system in the cytoplasmic membrane appears to harvest protonmotive force and transduce it in an unknown form to the outer membrane active transporters. Our long-term goal is to understand the mechanism of TonB-dependent energy transduction between the cytoplasmic and outer membranes of Escherichia coli. Mutations in tonB render many species significantly less virulent. Due to the role that iron availability plays in infectious disease, it is widely recognized the TonB system has potential as a novel target for the development of new antibiotics. Additional significance arises from our ability to contribute to a broad understanding of energy transduction mechanisms in general. There are currently no paradigms for TonB-dependent energy transduction and yet based on homologies, there are at least three such systems that transduce the pmf into useful work in virtually all Gram-negative bacteria. Besides the ExbB/D proteins, there are the TolQ/R proteins (required for OM integrity) and the MotA/B proteins (required for motility). Our results will therefore also contribute importantly to basic knowledge of the other energy transduction systems as broad principles emerge. As Specific Aims, we will address three fundamental problems in the TonB system: What are the determinants of TonB energization at the cytoplasmic membrane, what form of energy is transduced to the outer membrane transporters, and how does a transporter become energized by TonB? PUBLIC HEALTH RELEVANCE: Virtually all Gram negative bacteria use the TonB system to circumvent the limitations to iron acquisition posed by their outer membranes. Understanding of the molecular mechanism by which the TonB system energizes transport across the outer membranes will allow future development of new antibiotics that prevent iron uptake. The ability of pathogens to acquire iron is an important virulence determinant in infectious disease.